Objective:To investigate the antioxidant function of Mycoplasma pneumoniae MPN662 and analyze the key active sites, and to explore the role of MPN662 in the regulation of the production of reactive oxygen species (ROS) and superoxide dismutase (SOD) in THP-1 cells. Methods:pET28a(+ )- mpn662, recombinant mutant plasmids pET28a(+ )- mpn662-Ser 66 (the 66 th Cys was mutated to Ser) and pET28a(+ )- mpn662-Ala 66 (the 66 th Cys was mutated to Ala) were constructed, recombinant proteins rMPN662, rMPN662-Ser 66 and rMPN662-Ala 66 were expressed, identified, and purified. DTNB method was employed to analyze the MetO reduction activity of rMPN662 and recombinant mutant protein. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were applied to examine the transcription level of the mpn662 gene and the expression level of MPN662 protein after Mycoplasma pneumoniae were stimulated with different concentrations of hydrogen peroxide (H 2O 2) or tert-butyl hydroperoxide (t-BHP), respectively. Fluorescent probes (DCFH-DA) and the total SOD activity detection kit were used to test the levels of intracellular ROS and SOD in THP-1 cells, which were pretreated with rMPN662, and then stimulated by Mycoplasma pneumoniae lipid-associated membrane proteins (LAMPs). Results:Mycoplasma pneumoniae rMPN662 could reduce MetO to Met, and the enzyme activities of mutant protein were significantly lower than those of rMPN662 protein. mpn662 gene mRNA transcription level and MPN662 protein expression level were significantly increased in a dose-dependent manner when Mycoplasma pneumoniae was stimulated with H 2O 2 and t-BHP. Treatment with rMPN662 before THP-1 cells were exposed to LAMPs could decrease the level of ROS and increase the production of SOD. Conclusions:Mycoplasma pneumoniae MPN662 can reduce MetO to Met, and Cys66 is the key amino acid for this activity. MPN662 can decrease the release of ROS and increase the production of SOD in Mycoplasma pneumoniae LAMPs stimulated THP-1 cells.

Objective:To investigate the effects and regulation mechanism of lipid-associated membrane proteins (LAMPs) derived from Mycoplasma pneumoniae( Mp) on the expression of quinine oxidoreductase 1 (NQO-1) in human monocyte cell line THP-1 cells, and to know the effect of NQO-1 to interleukin 8 secretion in LAMPs stimulated cells, so as to better understand the regulation mechanism upon Mp infection. Methods:Mp were cultivated and the precipitate was collected to extract LAMPs. The cytotoxicity of LAMPs to THP-1 cells was analyzed by using CCK8 test. THP-1 cells were cultured in vitro with different concentrations of LAMPs for different times, and the expression of NQO-1 protein was detected by Western blot. Nrf2 siRNA was used to investigate the role of Nrf2 in NQO-1 expression in LAMPs induced cells, and NQO-1 inhibitor Diminutol was performed to test whether they blocked interleukin 8 (IL-8) secretion when treated with LAMPs in THP-1 cells. Results:LAMPs extracted from Mp had no cytotoxicity to THP-1 cells. The expression of NQO-1 protein in LAMPs-stimulated THP-1 cells showed a dose-dependent and time-dependent manner. The production of NQO-1 protein reached peaks when treated with 5.0 μg/ml or 7.5 μg/ml of LAMPs for 12 h. Silencing of Nrf2 by siRNA significantly decreased NQO-1 production, and blocking NQO-1 by Dim increased the level of IL-8 in LAMPs-stimulated cells. Conclusions:LAMPs derived from Mp induced the expression of NQO-1 protein in THP-1 cells via Nrf2, and NQO-1 can inhibit IL-8 secretion in LAMPs stimulated monocytes.

Objective: To investigate the influences of antibiotic-induced gut microbiota dysbiosis on Mycoplasma pneumoniae (Mp) airway infection. Methods: C57BL/6J mice were treated with vancomycin and gentamicin for 21 d by oral delivery and then intranasally infected with Mp. Quantitative real-time PCR (qPCR) was performed to detect five major phyla of gut microbiota in mouse fecal specimens before and after antibiotic treatment and the loads of Mp in lung tissues on 3 d and 7 d after infection. Pathological changes in lung tissues were evaluated with HE staining. IFN-γ and IL-4 secreted by spleen CD4⁺ T cells and CD8⁺ T cells were analyzed by flow cytometry. Mp-specific IgM and IgG in mouse serum samples were measured by indirect enzyme-linked immunosorbent assay (ELISA). Results: Vancomycin and gentamicin treatment significantly reduced the number of Bacteroidetes in mouse feces, but increased the amount of Firmicutes. Meanwhile, the numbers of δ, γ-Proteobacteria, Actinomycetes and Tenericutes also changed. These antibiotic-induced gut microbiota alterations in mice with Mp infection increased the loads of Mp in lung tissues and the pathological scores of lung tissue inflammation on 3 d and 7 d after infection, and reduced the number of IFN-γ-secreting spleen CD4⁺ T lymphocytes on 7 d. Conclusions Antibiotic-induced gut microbiota dysbiosis aggravated Mp airway infection.

Objective To investigate the prevalence of Mycoplasma pneumoniae ( Mp) infection in children in Hengyang from 2013 to 2016 and to analyze the p1 genotypes of the isolated Mp strains by using polymerase chain reaction-restriction fragment length polymorphism ( PCR-RFLP) , nested polymerase chain reaction (nPCR) and rapid-cycle polymerase chain reaction (Rapid-Cycle PCR).Methods Throat swab samples of children with acute respiratory tract infection were collected from four hospitals in Hengyang , Hu-nan Province from 2013 to 2016.Mp strains in these samples were identified by PCR amplification of the 16S rRNA gene.PCR-RFLP, nPCR and Rapid-Cycle PCR were performed for Mp p1 genotyping in order to fur-ther analyze the genotypes of Mp strains circulating in Hengyang .Results A total of 109 clinical strains of Mp were identified from the 984 throat swab samples .The sensitivities of PCR-RFLP and nPCR for genoty-ping MP strains were both 100％, while that of rapid-Cycle PCR was 98 .17％.All of the three methods showed 100％specificity for genotyping.Of all isolated Mp strains, 78.90％ were p1 gene type Ⅰ and 21.10％were p1 gene typeⅡ(t=93.239, P=0.01).From 2013 to 2016, the annual isolation rates of p1 gene type Ⅰ and type Ⅱ strains were 93.10％, 87.5％, 76.92％, 65.79％ and 6.90％, 12.5％, 23.08％, 34.21％, respectively.The rate of Mp p1 gene type Ⅰinfection decreased over year , while that of p1 gene type Ⅱinfection increased gradually .Conclusion PCR-RFLP, nPCR and rapid-Cycle PCR are reliable for genotyping of Mp p1 gene.The predominant genotype of Mp strains circulating in Hengyang is p 1 gene type Ⅰ, but the incidence of p 1 gene type Ⅱinfection gradually increases from 2013 to 2016 .

Objective To investigate the effects of lipid-associated membrane proteins ( LAMPs) derived from Mycoplasma pneumoniae ( M.pneumoniae) strains on the expression of heme oxygenase 1 ( HO-1) in a human monocyte cell line (THP-1).Methods THP-1 cells were in vitro cultured with different concentrations of LAMPs for different times.The cytotoxicity of LAMPs to THP-1 cells was analyzed by using lactate dehydrogenase ( LDH) releasing test.The expression of HO-1 at protein and mRNA levels were de-tected by Western blot and real-time RT-PCR, respectively.The enzymatic activity of HO-1 protein was ex-amined by colorimetric assay.THP-1 cells stimulated with PBS and LPS were set up as the negative and pos-itive controls, respectively.Results A significantly enhanced LDH releasing rate was observed in THP-1 cells treated with 10 μg/ml of LAMPs.The expression of HO-1 at protein and mRNA levels in THP-1 cells were induced by LAMPs in a dose-dependent and time-dependent manner.The highest level of HO-1 protein was detected in THP-1 cells treated with 5.0 μg/ml of LAMPs.The transcriptional levels of HO-1 induced by LAMPs were significantly elevated at 3 h, peaked at 9 h and were decreased at 12 h.The expression of HO-1 protein in THP-1 cells was enhanced after 8 h of treatment with LAMPs and a significant decrease was observed at 20 h after reaching peaks at 12 h and 16 h.The activity of HO-1 protein was significantly en-hanced along with the increased expression of HO-1 protein.Conclusion The LAMPs derived from M.pneumoniae strains induced the expression of HO-1 at mRNA and protein levels.Moreover, the enzyme activity of HO-1 protein was enhanced in LAMPs treated THP-1 cells.

Objective To investigate whether macrophage-activating lipopeptide-2 ( MALP-2) in-duces the expression of hemoxygenase-1 ( HO-1 ) in THP-1 cells and to further elucidate its possible regulatory mechanism for a better understanding of protective response upon mycoplasma infection .Methods THP-1 cells were cultured in vitro and stimulated by MALP-2 at different concentrations for 12 h.THP-1 cells were incubated with TLR 2 or TLR6 neutralizing antibodies , or transfected with their dominant negative plasmids to evaluate the effects of TLR 2 and TLR6 on HO-1 expression .Phosphorylation of Akt was detected by Western blot.PI3K inhibitor LY294002 was used to investigate the role of PI3K in HO-1 expression.Im-munofluorescence and electrophoretic mobility shift assay ( EMSA ) were performed to observe the nuclear translocation and DNA-binding activity of nuclear factor Nrf 2.Small interfering RNA ( siRNA) was used to silence the genes encoding Nrf2 and HO-1.Cobalt protoporphyrin (CoPP), an inducer of HO-1, was used to treat THP-1 cells.The expression of HO-1 was detected by Western blot .The secretion of TNF-αand IL-1βby THP-1 cells were measured by ELISA .Results MALP-2 induced the expression of HO-1 in THP-1 cells.However, the expression of HO-1 was inhibited by TLR2 and TLR6 neutralizing antibodies and expres-sion of their dominant negative plasmids .Moreover, PI3K pathway was activated by MALP-2, and with the use of PI3K inhibitor, the expression of HO-1 was decreased.The translocation of Nrf2 to the nucleus and itsDNA-binding activity were enhanced by MALP-2, but were inhibited by the treatment of PI3K inhibitor.Theexpression of HO-1 was significantly down-regulated upon the interference of Nrf2 gene expression withsiRNA.Silenced expression of HO-1 increased the level of TNF-αand IL-1β, while CoPP treatment decreasedthe secretion of MALP-2-induced cytokines.Conclusion MALP-2 might induce the expression ofHO-1 in THP-1 cells through TLR2,6/PI3K/Nrf2 pathways.The expression of HO-1 could negatively regulatethe hyper-secretion of cytokines.

Objective To investigate whether macrophage-activating lipopeptide-2 ( MALP-2) in-duces the expression of heme oxygenase-1 ( HO-1 ) in THP-1 cells and its possible mechanism .Methods Human monocyte cells THP-1 were cultured in vitro and then were incubated with various concentrations (0, 0.01, 0.1, 1.0 or 5.0 ng/ml) of MALP-2 for 16 h, or were stimulated by 5.0 ng/ml MALP-2 for different length of time (0 h, 4 h, 8 h, 12 h, 16 h or 24 h).The expression of HO-1 at mRNA and protein levels were detected by real-time PCR analysis and Western blot assay .The enzyme activity of HO-1 was detected by colorimetric analysis.THP-1 cells were pre-incubated with 30 μmol/L of SB203580, PD98059 and SP600125 for 30 min and then were cultured with 5.0 ng/ml MALP-2 for 16 h to investigate the role of mito-gen-activated protein kinases (MAPKs) signaling pathway in HO-1 production.After incubating THP-1 cells with 5.0 ng/ml MALP-2 for different periods of time, NF-E2-related factor 2 (Nrf2) protein was detected by Western blot assay to study the effects of Nrf2 pathway on MALP-2-induced HO-1 expression.Nrf2 and HO-1 proteins were measured by Western blot assay after transfecting THP-1 cells (1×106/well) with Nrf2 siRNA at a final concentration of 100 nmol/L.Results MALP-2 enhanced the expression of HO-1 at mRNA and protein levels as well as the enzyme activity of HO-1 in THP-1 cells in a concentration-dependent manner.The expression of HO-1 protein induced by MALP-2 was significantly inhibited by 30 μmol/L MAPKs specific inhibitors ( SB203580 , PD98059 and SP600125 ) .MALP-2 induced Nrf2 translocation at a concentration of 5.0 ng/ml.The expression of Nrf2 and HO-1 proteins were significantly decreased in Nrf 2 siRNA-transfected THP-1 cells.Conclusion MAPKs and Nrf2 signaling pathways were involved in the MALP-2 induced HO-1 expression .

Objective:To observe the molecular mechanism involved in expression of hemeoxygenase -1 (HO-1) induced by a macrophage-activating lipopeptide-2 (MALP-2).Methods:THP-1 cells were cultured in vitro and stimulated by MALP-2 for 12 h, expression of HO-1 was detected by Western blot .TLR2 and TLR6 neutralizing antibodies incubation , dominant negative plasmids transfection were used to assess the functional of TLR 2,6 in mediating HO-1 expression.Phosphorylation of c-Src and Akt were detec-ted by Western blot, and c-Src siRNA and PI3K inhibitor LY294002 were used to investigate the role of c-Src and PI3K in HO-1 ex-pression.Results:MALP-2 induced c-Src phosphorylation , and TLR2 and TLR6 neutralizing antibodies , or their dominant negatively plasmids could abrogate this effect .In addition, siRNA of c-Src could decrease the phosphorylation level of Akt , and the PI3K inhibi-tor could inhibit HO-1 expression.Conclusion: MALP-2 can induce THP-1 cells expression of HO-1 through TLR2,6/c-Src/PI3K pathways .

Objective To provide experimental evidence for the development of multi-epitope-baseded marker vaccines through investigating the humoral and cellular immune responses in BALB/c mice induced by the multiple antigen peptides (MAPs) with the mimic epitope.Methods Three types of MAPs in eight branched forms containing the mimic epitope of Mycoplasma genitalium adhesion protein (MgPa) were prepared using poly-lysine as the core matrix.The purity of MAPs was analyzed by reverse phase high performance liquid chromatography (RP-HPLC).The molecular weights of MAPs were characterized by Mass Spectrometry.The BALB/c mice were immunized intramuscularly for four times with single or mixed MAPs.The specific IgG antibody and the subtype of IgG antibody in serum of the immunized mice were detected by indirect ELISA.The proliferative responses of the spleen lymphocytes were detected using MTT assay.The ELISA were used to detect IFN-γ and IL-4 levels in the cultured supematant of spleen lymphocytes.Results The three types of MAPs containing the mimic epitopes were successfully prepared with high purity.They,could stimulate mice to produce specific IgG antibodies,of which,the major antibody isotype was Th1 immune response-associated IgG2a.Compared with the single MAP immunization group,the mixed-MAPs immunized mice produced more IgG,IgG1 and IgG2a antibody (P＜0.05).Furthermore,these MAPs could enhance the specific proliferation of spleen lymphocytes in immunized mice and induce the production of IFN-γ and IL-4.The levels of IFN-γand IL-4 in mixed-MAPs group were significantly higher than those of the single MAPs group (P＜0.01).Conclusion The three types of MAPs could induce strong specific cellular and humoral immune responses.The immunological competence of the mixed-MAPs was stronger than those of the single MAP.

Objective To determine the immunogenic and adhesive abilities of a segment (P1C protein) that located at the carboxy terminal region of P1 protein (1125 to 1395 amino acids).Methods A recombinant prokaryotic vector (pGEX6p-2/p1c) was constructed for P1C protein expression in E.coli BL21DE3.The expressed target recombinant protein (rP1C) was identified using SDS-PAGE and Western blot assay,and then extracted by GST-based affinity chromatography.The purified rP1C was used to immunize BALB/c mice to obtain rP1C-antiserum and titer of the antiserum was determined by ELISA.Immunoreactivity of the rP1C to the sera form M.pneumoniae-infected patients was detected using Western blot assay,while activity of the rP1C adhering to HeLa cells as well as adhesion blockage of the rP1C antiserum were detected using indirect immunofluorescence assays.Results The constructed prokaryotic expression system could efficiently express soluble rP1C with a relative molecular weight of 66×103.The antiserum from rP1Cimmunized mice showed an ELISA titer as high as 1:64 000.Both the M.pneumoniae-infected patients' sera and the mouse antiserum against rP1C could recognize as well as combine with the rP1C.rP1C could adhere to HeLa cells and the adhesion could be blocked by the mouse antiserum with an antiserum concentration-dependent manner.Conclusion P1C,a segment of M.pneumoniae P1 protein,possesses powerful immunogenicity and immunoreactivity and cell-adhered activity,indicating the protein segment can be used as an antigen candidate for developing vaccines and serological diagnostic methods of M.pneumoniae-induced diseases.